Adenovirus genomes are linear, double-stranded DNA molecules about 36 kilobase pairs long. Each extremity of the viral genome has a short sequence known as the inverted terminal repeat (or ITR), which is necessary for viral replication. The well-characterized molecular genetics of adenovirus render it an advantageous vector for gene transfer. The knowledge of the genetic organization of adenoviruses allows substitution of large fragments of viral DNA with foreign sequences. In addition, recombinant adenoviruses are structurally stable and no rearranged viruses are observed after extensive amplification.
Adenoviruses thus may be employed as delivery vehicles for introducing desired genes into eukaryotic cells, whereby the adenovirus delivers such genes to eukaryotic cells by binding cellular receptors, internalizing via coated pits, disrupting endosomes, and releasing particles to the cytoplasm followed by nuclear translocation and molecular expression of the adenovirus genetic program.
In general, in order to construct an adenoviral vector including a heterologous gene, or transgene, a plasmid is prepared which contains the transgene expression cassette and some adenoviral sequences, usually from the left end, or 5' end, of the virus. The plasmid then is co-transfected with DNA containing most of the adenoviral sequence, and homologous recombination occurs, yielding plaques on the adenoviral producer cells, such as, for example, 293 cells. The plaques are picked, amplified, screened, and scaled up further. Such process may take several months or more. If one desires to change the vector backbone, the process then involves further manipulation. If a single adenoviral gene is to be deleted from the adenoviral backbone, the first step is to generate a packaging cell that expresses the gene in trans. Next, a new plasmid is generated which encodes a portion of the adenoviral backbone with the adenoviral gene deleted. This plasmid is co-transfected with DNA encoding the remainder of the adenovirus. Homologous recombination then occurs, and adenoviral plaques are generated, screened, and scaled up, thus yielding a virus with an altered backbone. This virus then is used to generate a vector with a transgene by homologous recombination with the transgene-encoding plasmid as hereinabove described. The generation of such an adenoviral vector may take one year or more.